1. Field of the Invention
The present invention generally relates to surface treatment and particularly relates to methods and apparatus for treating surfaces such as biosensor surfaces by molecular chemisorption.
2. Discussion of Related Art
Production of biosensor arrays typically involves patterned deposition of biomolecules onto a surface. Sensitivity, reproducibility, and selectivity are significant aspects of biosensor quality. Sensitivity is typically achieved via a dense layer of chemisorbed capture molecules to efficiently capture target molecules. Reproducibility typically depends on highly reproducible anchoring chemistry and good quality patterning of capture molecules. Selectivity typically depends on high selectivity of target molecules and low non-selective adsorption of other molecules. The latter can limit utility of biosensors by accounting for 30% of the signal to be detected.
Bioconjugation involves linking molecules to form a complex having the combined properties of the individual components. Natural and synthetic compounds, and their activities, can be chemically combined to engineer substances having desired characteristics. For example, a protein bound to a target molecule in a complex mixture may be cross-linked with another molecule capable of being detected to form a traceable conjugate. The detection component provides visibility of the target component to produce a complex that can be localized, followed through various processes, or used for measurement.
Bioconjugation has affected many areas in the life sciences. Application of cross-linking reactions to creation of novel conjugates with particular activities has enabled the assay of minute quantities of substances for detection of cellular components and treatment of disease. The ability to chemically attach one molecule to another has produced a growing industry serving research, diagnostics, and therapeutic markets. A significant portion of biological assays is now performed using conjugates for interaction with specific analytes in solutions, cells, or tissues. An overview of conjugate molecules, reagent systems, and applications of bioconjugate techniques is given in G. T. Hermanson, “Bioconjugate Techniques”, Academic Press, San Diego, 1996.
Surfaces for use in biological environments are found in tools for molecular and cell biology such as substrates for Enzyme Linked Inmmunosorbent Assay (ELISA) in cell cultures, contact lenses, implanted prostheses, catheters, and containers for storage of proteins. Many such surfaces are quickly coated with a layer of proteins via spontaneous adsorption. Some have a beneficial effect. Others are detrimental. There is much interest in identifying biologically “inert” materials for resisting adsorption of proteins. A conventional surface treatment method for introducing such resistance involves coating the surface with poly(ethylene glycol) (PEG). See, for example J. M. Harris ed. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications, Plenum, New York, 1992. Further details of PEG are provided in Gombotz, W. R. et al., J. Biomed. Matter. Res. 15, 1547–1562 (1991). An alternative approach involves the pre-adsorption of bovine serum albumin. This approach however suffers from denaturation of the protein over time or exchange of the protein with other molecules. Therefore, this approach is unsuitable for application to biosensors for detecting presence of proteins by mass or primary amines. Self-assembled monolayers of short chain PEG oligomers (n=2−7) have also been shown to resist adsorption of proteins. See, for example Mrksich, M. and Whitesides, G. M., Annu. Rev. Biophys. Biomol. Struct. 25, 55–78 (1996). However, a protein attempting to adsorb may compress and desolvate PEG. These effects are both energetically disadvantageous. See, for example Jeon, S. I. and Andrade, J. D. “Protein surface interactions in the presence of polyethylene oxide: effect of protein size”, J. Coll. Interface Sci. 142, 159–166 (1991); and, Jeon, S. I., Lee, J. H., Adrade, J. D. and de Gennes, P. G., “Protein surface interactions in the presence of polyethyleneoxide: simplified theory”, J. Coll. Interface Sci. 142, 149–158 (1991)).
Biosensor surfaces are typically formed from borosilicate glass. Various conventional schemes for attaching capture molecules, such as antibodies or DNA oligomers, to a biosensor surface will now be described.
Referring to FIG. 1A, in one conventional scheme, direct chemisorption or physisorption binds capture molecules 10 to the surface 20. The surface 20 is functionalized with relatively short linker molecules 30. For example, the surface 20 may be amine-functionalized. The linkers 30 immobilize the capture molecules 10 on the surface 20. Unwanted molecules also present on the surface 20 can be typically removed by stringent washing to optimize selectivity of the biosensor. However, washing can remove physisorbed molecules. Chemisorbed molecules are more resistant to washing. Referring to FIG. 1B, this scheme leaves many linkers 30 exposed. This leads to much nonspecific chemisorption or physisorption of other molecules 40. This reduces the selectivity of the biosensor. The ratio of molecules bound by specific interaction to molecules bound by nonspecific interaction is reduced. Although the selectivity of the capture molecules 10 may be high in solution, many detection methods are convoluted by presence of other molecules 40. Additionally, direct physisorption or chemisorption of the capture molecules 10 limits molecular mobility. Few capture molecules 10 have full functionality. The sensitivity of the capture molecules 10 is thus reduced. Binding efficiency is usually reduced where the capture molecules 10 are for binding assays. The affinity constant and binding kinetics vary in dependence on the orientation of chemisorption. This results in less target molecules 70 such as antigens being bound to the surface 20. Also, there is higher binding variability between different surfaces. If the detection scheme employed cannot distinguish between the target molecules 70 and the other molecules 40, the effective specificity of the biosensor is significantly reduced. This is common in both label free and labelled detection schemes. Stringent washing does not usually correct this problem because cooperative effects during nonspecific binding are virtually irreversible.
Referring to FIG. 1C, in another conventional scheme, the capture molecules 10 are chemisorbed to the surface 20 through spacer molecules 50. As indicated earlier, chemisorption of capture molecules 10 allows more stringent washing procedures, thus reducing nonspecifically physisorbed molecules. The spacers 50 are longer than the linkers 30 herein before described with reference to FIG. 1A. The spacers 50 tether the capture molecules 10 to the surface 20. However, the spacers 50 also allow limited movement of the capture molecules 10 relative to the surface 20. This allows the capture molecules 10 increased activity. The mobility and thus functionality of capture molecules 10 is improved. Binding efficiency of the capture molecules 10 chemisorbed through spacers 50 is thus increased. However, referring to FIG. 1D, the exposed surface 20 and the spacers 50 allow nonspecific binding of other molecules 40. Nonspecific binding can occur in roughly the same amount as in the FIG. 1B arrangement.
Referring to FIG. 1E, in a modification of the FIG. 1C scheme, the capture molecules 10 anchored to the surface 20 through spacers 50 in a sea of biocompatible molecules 60. The biocompatible molecules 60 are resistant to nonspecific adsorption of the other molecules 40. This improves the effective specificity of the surface 20. Referring to FIG. 1F, nonspecific binding is reduced because the surface 20 is less exposed. Only the spacers 50 and the capture molecules 10 offer sites for nonspecific binding of other molecules 40. Stringent washing can improve specificity further by removing more nonspecifically bound molecules 40 than specifically bound molecules 10.
The susceptibility of biosensors to nonspecific adsorption also depends on labeling and detection schemes employed. For example, sandwich ELISA assays are less susceptible because the signal measured depends only on the number of target antigen molecules 10 and the specificity with which labeling antibodies bind to the surface 20. This approach is inert because it involves a dual selective detection. However, label-free detection schemes are more susceptible to other molecules 40 adsorbing nonspecifically to the surface 20. These schemes measure the protein/DNA present on the surface 20 by mass or refractive index. More control over nonspecific adsorption is needed than in sandwich ELISA biosensors. Control over nonspecific adsorption and nonspecific signal generation is also important where bound molecules are detected by chemical labeling techniques. Such control is particularly desirable when chemical labeling exhibits cross-reactivity with chemical groups involved in the chemisorption. A barrier can be added to prevent access to the groups. However, BSA blocking of nonspecific adsorption is not usually possible because BSA produces a signal and reduces the “effective” selectivity of the biosensor.
Nucleic acid aptamers are useful for biosensor production because they can form three dimensional structures. Aptamers can also bind a range of target molecules with acceptable affinity and specificity. Also, aptamers can function in a similar manner to protein molecules. For example, aptamers can change in structure due to ligand-binding. There are many different receptors that are useful in biosensor arrays. However, aptamers are especially useful for several reasons. One reason is that aptamers can be more easily engineered than antibodies. Following selection, aptamers can be reduced to 30–60 nucleotide residue core sequences without reducing binding function. Another reason is that modifications such as fluorescent reporters can be easily introduced by chemical synthesis. Yet another reason is that aptamer structure is mainly a function of Watson-Crick base pairing. Secondary structural interactions allow aptamers to be more easily converted to receptors than antibodies.
Referring to FIG. 2A, in a conventional process for producing a biosensor as herein before described with reference to FIG. 1F, capture molecules in the form of amine-functionalized aptamers 120 are crosslinked onto a glass surface 20. The surface is first functionalized with APTS (3-aminopropyl triethoxysilane) 100. The cross-linking is then performed by spacers in the form of bifunctional succinimide crosslinkers (BS3) 110.
Following such chemisorption of capture molecules 10, the remaining crosslinking spacers 50 and amine surface 20 are blocked with a single functionalized succinimide to cover the amines, and ethanolamine to saturate unreacted succinimide groups. Any remaining surface areas may be treated with PEG. These post-chemisorption steps reduce nonspecific protein absorption. However, as herein before indicated, spaces between the capture molecules 10 and the spacers 50 can still react. These spaces accept nonspecific protein adsorption. BS3 spacers are too short for many proteins and pose problems as herein before described with reference to FIG. 1A. In addition, the functionalizing, cross-linking, and blocking steps each involve exposure of the surface 20 to a different environment in a different bath. This process is laborious, time consuming, and wasteful of raw materials.
It would be desirable to provide a method for effecting chemisorption of capture molecules with improved activity, accessibility, capacity, and specificity of the capture molecules. In particular, it would be desirable to provide a method for biosensor fabrication that: irreversibly attaches capture molecules to the biosensor surface; provides sufficient mobility and accessibility for capture molecules to remain functional; and, minimizes nonspecific adsorption of target antigens or other molecules. It would also be desirable to provide a method for fabricating biosensors which is less laborious, less time consuming, and less wasteful of materials.